Assessment of the anti-pathogenic effects of condensed tannin extracts using scanning electron microscopy.

Two different types of condensed tannins (CTs), which were extracted and purified from tilia (Tilia L.) and black locust (Robinia pseudoacacia), were studied and tested against two kinds of bacteria, including Gram-negative and Gram-positive, avian pathogenic E. coli (APEC) and Staphylococcus epidermidis (S. epidermidis) respectively, by minimal bactericidal concentrations (MBCs) and scanning electron microscopy (SEM). Both CT extracts were significantly effective (p ≤ 0.05) at MBCs of 5-10 mg CT/ml against APEC (Gram-negative), and at 1.25-5 mg CT/ml on S. epidermidis (Gram-positive). This indicated that the CTs were more potent against the Gram-positive than the Gram-negative bacteria. Further, SEM revealed that CTs caused mainly morphological deformations of the bacterial cells and some conjoined cell growth.

HPLC coupled with quadrupole time of flight tandem mass spectrometry for analysis of glycosylated components from the fresh flowers of two congeneric species: Robinia hispida L. and Robinia pseudoacacia L.

Developing methods for the systematic and rapid identification of the chemical compositions of fresh plant tissues has long attracted the attention of phytochemists and pharmacologists. In the present study, based on highly efficient sample pretreatment and high-throughput analysis of high-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry data using molecular networks, a method was developed for systematically analyzing the chemical constituents of the fresh flowers of Robinia hispida L. and Robina pseudoacacia L., two congeneric ornamental species that lack prior consideration. A total of 44 glycosylated structures were characterized. And on the basis of establishing of the fragmentation pathways of 11 known flavonoid glycosides, together with the molecular networking analysis, 18 other ions of flavonoid glycosides in five classes were clustered. Moreover, 15 soyasaponins/triterpenoid glycosides were tentatively identified by comparison of their tandem mass spectrometry characteristic ions with those reported in the literature or the online Global Natural Product Social Molecular Networking database. The water extracts were separated by flash chromatography, which resulted in the discovery of one new compound, named rohispidascopolin, along with five known entities. The pharmacological targets were predicted by SwissTargetPrediction.

Glandular trichomes of Robinia viscosa Vent. var. hartwigii (Koehne) Ashe (Faboideae, Fabaceae)-morphology, histochemistry and ultrastructure.

MAIN CONCLUSION: Permanent glandular trichomes of Robinia viscosa var. hartwigii produce viscous secretion containing several secondary metabolites, as lipids, mucilage, flavonoids, proteins and alkaloids. Robinia viscosa var. hartwigii (Hartweg's locust) is an ornamental tree with high apicultural value. It can be planted in urban greenery and in degraded areas. The shoots, leaves, and inflorescences of this plant are equipped with numerous persistent glandular trichomes producing sticky secretion. The distribution, origin, development, morphology, anatomy, and ultrastructure of glandular trichomes of Hartweg's locust flowers as well as the localisation and composition of their secretory products were investigated for the first time. To this end, light, scanning, and transmission electron microscopy combined with histochemical and fluorescence techniques were used. The massive glandular trichomes differing in the distribution, length, and stage of development were built of a multicellular and multiseriate stalk and a multicellular head. The secretory cells in the stalk and head had large nuclei with nucleoli, numerous chloroplasts with thylakoids and starch grains, mitochondria, endoplasmic reticulum profiles, Golgi apparatus, vesicles, and multivesicular bodies. Many vacuoles contained phenolic compounds dissolved or forming various condensed deposits. The secretion components were transported through symplast elements, and the granulocrine and eccrine modes of nectar secretion were observed. The secretion was accumulated in the subcuticular space at the trichome apex and released through a pore in the cuticle. Histochemical and fluorescence assays showed that the trichomes and secretion contained lipophilic and polyphenol compounds, polysaccharides, proteins, and alkaloids. We suggest that these metabolites may serve an important function in protection of plants against biotic stress conditions and may also be a source of phytopharmaceuticals in the future.

Low-cost field production of biochars and their properties.

Biochar has been intensively investigated for carbon sequestration, soil fertility enhancement, and immobilization of heavy metals and organic pollutants. Large-scale use of biochar in agricultural production and environmental remediation, however, has been constrained by its high cost. Here, we demonstrated the production of low-cost biochar ($20/ton) in the field from Robinia pseudoacacia biowaste via a combined aerobic and oxygen-limited carbonization process and a fire-water-coupled method. It involved aerobic combustion at the outer side of biomass, oxygen-limited pyrolysis in the inner core of biomass, and the termination of the carbonization by water spray. The properties of biochar thus produced were greatly affected by exposure time (the gap between a burning char fell to the ground and being extinguished by water spray). Biochar formed by zero exposure time showed a larger specific surface area (155.77 m2/g), a higher carbon content (67.45%), a lower ash content (15.38%), and a higher content of carboxyl and phenolic-hydroxyl groups (1.74 and 0.86 mol/kg, respectively) than biochars formed with longer exposure times (5-30 min). Fourier-transform infrared spectroscopic (FTIR) spectra indicated that oxygen-containing functional groups of biochar played a role in Cd and oxytetracycline sorption though a quantitative relationship could not be established as the relative contribution of carbon and ash moieties of biochar to the sorption was unknown. Outcomes from this research provide an option for inexpensive production of biochar to support its use as a soil amendment in developing countries.

Development of a Female-Targeted Lure for the Box Tree Moth Cydalima perspectalis (Lepidoptera: Crambidae): a Preliminary Report.

The box tree moth, Cydalima perspectalis, is an invasive pest in Europe causing damage on Buxus species. In this study, we aimed to develop a "bisexual" lure to attract both female and male moths. Based on a previous screening bioassay we tested methyl salicylate, phenylacetaldehyde and eugenol as potential attractants in different combinations. The trapping results showed that both binary and ternary blends attracted male and female moths. Catches with these blends were comparable to catches with the synthetic pheromone. Subsequently we carried out single sensillum recordings, which proved the peripheral detection of the above-mentioned compounds on male and female antennae. To identify synergistic flower volatiles, which can be also attractive and can increase the trap capture, we collected flower headspace volatiles from 12 different flowering plant species. Several components of the floral scents evoked good responses from antennae of both females and males in gas chromatography-electroantennographic detection. The most active components were tentatively identified by gas chromatography coupled mass spectrometry as benzaldehyde, cis-ß-ocimene, (±)-linalool and phenethyl alcohol. These selected compounds in combination did not increase significantly the trap capture compared to the methyl salicylate- phenyacetaldehyde blend. Based on these results we discovered the first attractive blend, which was able to attract both adult male and female C. perspectalis in field conditions. These results will yield a good basis for the optimization and development of a practically usable bisexual lure against this invasive pest.

Volatile-Compound Fingerprinting by Headspace-Gas-Chromatography Ion-Mobility Spectrometry (HS-GC-IMS) as a Benchtop Alternative to 1H NMR Profiling for Assessment of the Authenticity of Honey.

This work describes a simple approach for the untargeted profiling of volatile compounds for the authentication of the botanical origins of honey based on resolution-optimized HS-GC-IMS combined with optimized chemometric techniques, namely PCA, LDA, and kNN. A direct comparison of the PCA-LDA models between the HS-GC-IMS and 1H NMR data demonstrated that HS-GC-IMS profiling could be used as a complementary tool to NMR-based profiling of honey samples. Whereas NMR profiling still requires comparatively precise sample preparation, pH adjustment in particular, HS-GC-IMS fingerprinting may be considered an alternative approach for a truly fully automatable, cost-efficient, and in particular highly sensitive method. It was demonstrated that all tested honey samples could be distinguished on the basis of their botanical origins. Loading plots revealed the volatile compounds responsible for the differences among the monofloral honeys. The HS-GC-IMS-based PCA-LDA model was composed of two linear functions of discrimination and 10 selected PCs that discriminated canola, acacia, and honeydew honeys with a predictive accuracy of 98.6%. Application of the LDA model to an external test set of 10 authentic honeys clearly proved the high predictive ability of the model by correctly classifying them into three variety groups with 100% correct classifications. The constructed model presents a simple and efficient method of analysis and may serve as a basis for the authentication of other food types.

From Robinia pseudoacacia L. nectar to Acacia monofloral honey: biochemical changes and variation of biological properties.

BACKGROUND: Robinia pseudoacacia L. nectar and its derivative monofloral honey were systematically compared in this study, to understand how much the starting solution reflected the final product, after re-elaboration by Apis mellifera ligustica Spinola. RESULTS: Subjected to dehydration in the hive, nectar changed in its water and sugar content when transformed into honey, as physicochemical and gas chromatographic-mass spectrometric analyses revealed. Spectrophotometric measurements and characterization by high-performance liquid chromatography-diode array detection of 18 plant molecules demonstrated honey to be richer than nectar in secondary metabolites. For the first time, the hypothesis of the existence of a nectar redox cycle in R. pseudoacacia was reported, as previously described for Nicotiana sp., based on 1D-protein profiles, western blot analysis and detection of H2 O2 and ascorbate. The bioactivity of both matrices was also investigated. Antiradical in vitro tests showed that Acacia honey was more antioxidant than nectar, which was even able to induce oxidative stress directly in a eukaryotic cell system. Antimicrobial assays demonstrated that nectar was bacteriostatic, due to H2 O2 activity, whereas honey was even bactericidal. CONCLUSION: All these data support the ecological role of nectar and honey in nature: protection of the gynoecium from pathogens and preservation from degradative processes, respectively. © 2018 Society of Chemical Industry.

The Taishan Robinia pseudoacacia polysaccharides enhance immune effects of rabbit haemorrhagic disease virus inactivated vaccines.

Robinia pseudoacacia flower, a common component in traditional Chinese medicine, has long been well-known for its high pharmaceutical value. This study aimed to assess the immunopotentiating effects of Taishan Robinia Pseudoacacia polysaccharides (TRPPS) in rabbits inoculated with a rabbit haemorrhagic disease virus (RHDV) inactivated vaccine. The rabbits were administered with the RHDV vaccine in conjunction with varying concentrations of TRPPS, and their blood samples were collected at different time points to analyze the ratio and number of blood lymphocytes. In addition, sera were prepared and analyzed to determine the overall antibody titer and the level of IL-2, a cytokine commonly used as an indicator of immune activity. The various TRPPS-supplemented vaccines were shown to be more effective in enhancing the immune functions of the inoculated rabbits compared to their polysaccharide-free counterpart, with 200 mg/mL of TRPPS exhibiting the most pronounced benefits that were comparable to those of propolis. In addition, the TRPPS-supplemented RHDV inactivated vaccines could significantly improve the survival rates of the immunized rabbits against RHDV infection. Our studies offered convincing experimental evidence for the development of TRPPS as a new type of plant-derived immunopotentiator.

Enzymatic activities for lignin monomer intermediates highlight the biosynthetic pathway of syringyl monomers in Robinia pseudoacacia.

Most of the known 4-coumarate:coenzyme A ligase (4CL) isoforms lack CoA-ligation activity for sinapic acid. Therefore, there is some doubt as to whether sinapic acid contributes to sinapyl alcohol biosynthesis. In this study, we characterized the enzyme activity of a protein mixture extracted from the developing xylem of Robinia pseudoacacia. The crude protein mixture contained at least two 4CLs with sinapic acid 4-CoA ligation activity. The crude enzyme preparation displayed negligible sinapaldehyde dehydrogenase activity, but showed ferulic acid 5-hydroxylation activity and 5-hydroxyferulic acid O-methyltransferase activity; these activities were retained in the presence of competitive substrates (coniferaldehyde and 5-hydroxyconiferaldehyde, respectively). 5-Hydroxyferulic acid and sinapic acid accumulated in the developing xylem of R. pseudoacacia, suggesting, in part at least, sinapic acid is a sinapyl alcohol precursor in this species.

Characterisation of Antimicrobial Properties of Extracts of Selected Medicinal Plants.

The scope of the experiments included analysis of the antimicrobial activity of ethanolic, methanolic and aqueous extracts against bacterial and fungal cultures and determination of the minimum inhibitory concentration of plant extracts tested microbial growth. Analysis of the antifungal and antibacterial activity was carried out by the disc diffusion method using paper discs. In the experiment 11 species of microorganisms - 8 bacterial and 3 fungal strains were used. The highest antimicrobial activity against the tested strains was demonstrated by black elder (Sambucus nigra L.), black locust (Robinia pseudoacacia L.) and lingonberry (Vaccinium vitis-idaea L.) extracts. The study showed the diverse morphological activity of specific parts of elderberry and quince, which is the effect of different polyphenolic profile of these plants. The yeast Saccharomyces cerevisiae, Escherichia coli, Pseudomonas putida and Bacillus subtilis showed the highest sensitivity to the effect of extracts of the analysed plants. As a positive control three antibiotics - amphotericin B, vancomycin and amoxicillin with clavulanic acid were used.

Hydroponic screening of black locust families for heavy metal tolerance and accumulation.

Present work examines phytoextraction potential of four black locust families (half-sibs 54, 56, 115, and 135) grown hydroponically. Plants were treated with 6 ppm of cadmium (Cd), 100 ppm of nickel (Ni), and 40 ppm of lead (Pb) added in Hoagland nutrient solution, accompanying with simultaneously applied all three metals. Responses to metals exposure among families were different, ranging from severe to slight reduction of root and shoot biomass production of treated plants. Calculated tolerance indices are indicating tested families as highly tolerant (Ti > 60). Family 135 had the lowest tolerance index, pointing that it was highly susceptible to applied metals. Comparing photosynthetic activities of tested families it has been noticed that they were highly sensitive to stress induced by heavy metals. Net photosynthetic rate of nickel treated plants was the most affected by applied concentration. Cadmium and nickel concentrations in stems and leaves of black locust families exceeded 100 mg Cd kg(-1) and 1000 mg Ni kg(-1), in both single and multipollution context. On the contrary, accumulation of lead in above ground biomass was highly affected by multipollution treatment. Tf and BCF significantly varied between investigated treatments and families of black locust. Concerning obtained results of heavy metals accumulation and tolerance of black locust families can be concluded that tested families might be a promising tool for phytoextraction purposes, but it takes to be further confirmed in field trials.

Evaluation of genotoxic variations in plant model systems in a case of metal stressors.

This study was designed to assess the effects of long term, high metal exposition (cadmium, lead, copper, nickel and zinc) on DNA damage in four plant model systems [Taraxacum officinale (Asteraceae), Matricaria recutita L. (Asteraceae), Robinia pseudoacacia L. (Fabaceae), and Urtica dioica (Urticaceae)]. DNA stability was investigated by a Random Amplified Polymorphic DNA (RAPD) technique. Agarose-gel electrophoresis revealed total of 37 bands with different molecular weights ranging from 1250 to 5000 bp. It generated distinctive polymorphism value of 72.97% (27 bands) total in four plant species investigated. The dendrogram constructed using NTSYSpc programme showed that there is grouping in separate clusters of the same plant model collected from two different areas (metal-exposed and control samples). The study concluded that the long term metal-exposing periods had genotoxic stress on macromolecules of plant model systems investigated and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of plants to metal stressors.

Structural basis for carbohydrate binding properties of a plant chitinase-like agglutinin with conserved catalytic machinery.

A new chitinase-like agglutinin, RobpsCRA, related to family GH18 chitinases, has previously been identified in black locust (Robinia pseudoacacia) bark. The crystal structure of RobpsCRA at 1.85Å resolution reveals unusual molecular determinants responsible for the lack of its ancestral chitinase activity. Unlike other chitinase-like proteins, which lack chitinase catalytic residues, RobpsCRA has conserved its catalytic machinery. However, concerted rearrangements of loop regions coupled to non-conservative substitutions of aromatic residues central to the chitin-binding groove explain the lack of hydrolytic activity against chitin and the switch toward recognition of high-mannose type N-glycans. Identification of close homologs in flowering plants with conservation of sequence motifs associated to the structural adaptations seen in RobpsCRA defines an emerging class of agglutinins, as emphasized by a phylogenetic analysis, that are likely to share a similar carbohydrate binding specificity for high-mannose type N-glycans. This study illustrates the recent evolution and molecular adaptation of a versatile TIM-barrel scaffold within the ancestral GH18 family.

Bactericidal effect of extracts and metabolites of Robinia pseudoacacia L. on Streptococcus mutans and Porphyromonas gingivalis causing dental plaque and periodontal inflammatory diseases.

The mouth cavity hosts many types of anaerobic bacteria, including Streptococcus mutans and Porphyromonas gingivalis, which cause periodontal inflammatory diseases and dental caries. The present study was conducted to evaluate the antibacterial potential of extracts of Robinia pseudoacacia and its different fractions, as well as some of its natural compounds against oral pathogens and a nonpathogenic reference bacteria, Escherichia coli. The antibacterial activity of the crude extract and the solvent fractions (hexane, chloroform, ethyl acetate and butanol) of R. pseudoacacia were evaluated against S. mutans, P. gingivalis and E. coli DH5α by standard micro-assay procedure using conventional sterile polystyrene microplates. The results showed that the crude extract was more active against P. gingivalis (100% growth inhibition) than against S. mutans (73% growth inhibition) at 1.8 mg/mL. The chloroform and hexane fractions were active against P. gingivalis, with 91 and 97% growth inhibition, respectively, at 0.2 mg/mL. None of seven natural compounds found in R. pseudoacacia exerted an antibacterial effect on P. gingivalis; however, fisetin and myricetin at 8 µg/mL inhibited the growth of S. mutans by 81% and 86%, respectively. The crude extract of R. pseudoacacia possesses bioactive compounds that could completely control the growth of P. gingivalis. The antibiotic activities of the hexane and chloroform fractions suggest that the active compounds are hydrophobic in nature. The results indicate the effectiveness of the plant in clinical applications for the treatment of dental plaque and periodontal inflammatory diseases and its potential use as disinfectant for various surgical and orthodontic appliances.

Bioaccumulation of heavy metals by the leaves of Robinia pseudoacacia as a bioindicator tree in industrial zones.

The study is a part of research project on using short rotation plantations in biomass production. The aim of this investigation was to determine the accumulation characteristics of Robinia pseudoacacia leaves as a biomonitor of heavy-metal pollution in the industrial regions in Bulgaria. The study was carried out in 25-year-old plantations located in close vicinity of industrial area Devnia, Eastern Bulgaria. Devnia is a zone, highly contaminated by cement factories, nitrogen fertilizers and polyvinylchloride factories. Controls were similarly aged plantations on a background area 15 km away from the emission sources. The concentration of nutrients and heavy metals in the leaves of damaged and control plants were investigated. Decreased levels of total nitrogen (6%), total phosphorous (11%), potassium (36%) and magnesium (3%) were detected in September for polluted trees as compared to control trees. Lead content (30.7 mg kg(-1)) was 1.38 times higher as compared to control, whereas accumulation of zinc (19.0 mg kg2) about 1.37 times more than control. An excessive accumulation of copper in the leaves collected from the polluted area (17.2 mg kg(-1)) was 2.15 times higher than control. The concentrations of Pb, Zn and Cu in the contaminated soil under black locust plantation were 38.2, 77.4 and 101.3 mg kg(-1), respectively. Our results showed that the leaves of R. pseudoacacia accumulated Pb, Zn and Cu in parallelity with their increase in the contaminated soil. R. pseudoacacia may be considered as a good biomonitor of soil pollution especially with Cu, and at lower extent for Pb and Zn in the industrial region of Devnia.

From flower to honey bouquet: possible markers for the botanical origin of Robinia honey.

Flowers are complex structures devoted to pollinator attraction, through visual as well as chemical signals. As bees collect nectar on flowers to produce honey, some aspects of floral chemistry are transferred to honey, making chemical markers an important technique to identify the botanical and geographical origins of honey. We applied a new approach that considers the simultaneous analysis of different floral parts (petals, stamens + pistils, calyxes + nectarines, and nectar) and the corresponding unifloral honey. We collected fresh flowers of Robinia pseudoacacia L. (black locust), selected five samples of Robinia honey from different geographical origins, applied SPME-GC/MS for volatile analyses, and defined the chemical contribution added by different floral parts to the honey final bouquet. Our results show that honey blends products from nectar as well as other flower parts. Comparing honey and flower profiles, we detected compounds coming directly from flower parts but not present in the nectar, such as hotrienol and ß-pinene. These may turn out to be of special interest when selecting floral markers for the botanical origin of honey.

Total synthesis and dual PPARα/γ agonist effects of amorphastilbol and its synthetic derivatives.

Amorphastilbol (APH-1), isolated from a Robinia pseudoacacia var. umbraculifer [corrected] seed extract, is a biologically interesting natural trans-stilbene compound with dual peroxisome proliferator-activated receptor (PPAR) α/γ agonist activity. After total synthesis of APH-1 and its derivatives by Pd-catalyzed Suzuki-Miyaura cross-coupling of a common (E)-styryl bromide intermediate and various aromatic trifluoroborate compounds, we biologically evaluated APH-2-APH-12 for PPAR agonist activity. APH-4 and APH-11 were effective PPARα/γ transcriptional activators, compared with APH-1. Therefore, we suggest that APH-4 and APH-11 are novel dual PPARα/γ agonists and are potentially useful for treating type 2 diabetes by enhancing glucose and lipid metabolism.

Quantification of canavanine, 2-aminoethanol, and cyanamide in Aphis craccivora and its host plants, Robinia pseudoacacia and Vicia angustifolia: effects of these compounds on larval survivorship of Harmonia axyridis.

The cowpea aphid Aphis craccivora that infests the black locust Robinia pseudoacacia shows toxicity to its predator, the multicolored Asian ladybird beetle, Harmonia axyridis. In contrast, the same aphid species that infests the common vetch, Vicia angustifolia, is suitable prey for H. axyridis larvae. Previously, it was reported that the toxicity of A. craccivora infesting R. pseudoacacia was due to canavanine and 2-aminoethanol, but there was some doubt about the toxicity of these compounds and their concentrations in the aphids. In the present study, we determined the concentrations of cyanamide, canavanine, and 2-aminoethanol in A. craccivora infesting the two host plants. In the extracts of A. craccivora that infested either of the host plants, canavanine was undetectable, and 2-aminoethanol was detected at the concentration of 3.0-4.0 µg/g fresh weight. Cyanamide was detected in the extract of A. craccivora that infested R. pseudoacacia (7.7 µg/g fresh weight) but not in that infesting V. angustifolia. The toxicity of canavanine, 2-aminoethanol, and cyanamide was evaluated against H. axyridis larvae in a bioassay by using an artificial diet containing these compounds at various concentrations. Cyanamide exhibited 10-100 times stronger toxicity than canavanine and 2-aminoethanol. These results indicate that the toxicity is at least partly due to cyanamide, which is present in the toxic A. craccivora that infests R. pseudoacacia but absent from the non-toxic A. craccivora that infests V. angustifolia.

Quantification of cyanamide in young seedlings of Vicia species, Lens culinaris, and Robinia pseudo-acacia by gas chromatography-mass spectrometry.

We quantified the cyanamide content of young leaves of nine Vicia species, Lens culinaris, and Robinia pseudo-acacia using a modified analytical procedure that made it possible to measure the cyanamide content of a single leaf. Recent molecular phylogenetic analysis suggests that cyanamide is present in V. benghalensis, which is placed in a monophyletic group with cyanamide-biosynthesizing plants, V. villosa and V. cracca; this suggestion was verified.

Assessment of airborne heavy metal pollution using plant parts and topsoil.

Robinia pseudoacacia L. (Fabaceae) was evaluated as a possible bioindicator of airborne heavy metal pollution, which originates from mining and pyrometallurgical copper production in Bor (Eastern Serbia). Concentrations of Cu, Zn, Pb, Cd, As and Hg were determined in different plant organs (washed/unwashed leaves, branches, roots) and topsoil of R. pseudoacacia by ICP-AES and by AAS. Sampling was carried out during 2008 at ten selected sites distributed in five zones with different levels of pollution. Concentrations of Pb, Cd and Hg did not exceeded the maximum allowed concentration (MAC) in soils at any of the sampling sites. Cu and As were present only at two sites within the MAC, whereas Zn exceeded the MAC at two sampling sites. Although present in the soil, As, Cd and Hg were below limit of detection in all parts of R. pseudoacacia. The rest of the studied elements, collected at the sites closest to the copper smelter or in the directions of the prevailing winds, were found to be at high levels. The higest Cu and Zn concentrations were detected in branches of R. pseudoacacia at the site Krivelj in the rural zone (6418.2±355.4 mg kg⁻¹ and 4699.8±320.8 mg kg⁻¹, respectively). Pb was present in similar amounts in all parts of R. pseudoacacia in the concentration ranging from 4.9 ± 0.3 mg kg⁻¹ (in washed leaves, at tourist zone) to 66.9±5.3 mg kg⁻¹ (in roots, at urban-industrial zone). According to the mobility ratio, leaves and branches of R. pseudoacacia acted as excluders of Cu, Zn and Pb, except for the branches which acted as indicators of Zn. Although As is present in high concentrations in the air and topsoil of the examined area, results show that R. pseudoacacia is not a suitable indicator of environmental pollution with As.